Thursday, August 18, 2011

BLOOD CULTURE Automation

Culture of patient’s blood is one of the most important investigations in clinical microbiology.
Uses
Blood culture is requested mainly in two clinical situations:
(1) where the possibility of septicemia or bacteriaemia is suggested by the presence of fever, shock or other symptoms occurring in association with a known or suspected local infection such as sepsis in a surgical wound, puerperal sepsis, pneumonia, meningitis, osteomyelitis or endocarditis.
(2) where it is one of the procedures required in the investigation of a fever difficult to diagnose because of the absence of signs of a specific infection or local infective lesion, i.e. a pyrexia of unknown origin (PUO).
Collection of sample.
To minimize difficulty in interpretation, every care must be taken to prevent contamination of the specimen during its collection from the patient and its in the laboratory. They should be instructed how to disinfect the skin over the vein, to use a fresh sterile syringe for the venepuncture and to replace the needle used for the venpuncture with a fresh sterile needle before inoculating the blood into the culture bottles They should hold the needle by its butt, not shaft, either with sterile forceps or with the fingers covered with a dry sterile rubber glove, and should take care to avoid contaminating themselves or the outside of the culture bottles with potentially infective blood.
They should also be warned that contamination is very likely. if the sample is collected from an indwelling peripheral venous catheter or a long central venous line instead of from a fresh venepuncture. In some cases such lines themselves become colonized with skin commensal bacteria which are then seeded into the circulation.
Culture Method
It is necessary to culture at least several milliliters of blood diluted I in 5 to 1 in 10 in a culture medium.
This is conveniently done b) inoculating 5-10 ml blood into 50 ml liquid medium in a bottle of 90-125ml capacity. To maximize the chances of detecting scanty bacteria, a larger volume, e.g. 10-20 ml, of blood should be collected and half of it should be inoculated into each of a ‘set’ of two such culture bottles. The use of two bottles has several further advantages: they may contain different media or be incubated different atmospheres and scanty contaminants are likely to grow in only one of them.
The bottles of sterile medium should be tightly closed with a perforated aluminum or plastic cap over an entire rubber washer, and these should be covered and kept sterile till the moment of inocu¬lation by a metal foil cover. After removal of the foil, the blood is inoculated into the bottle by inserting the syringe needle through the rubber washer, the bottle remaining unopened and unexposed to the entry of airborne dust. Instructions for the collection of the sample and its introduction into the bottle should be given on a printed label attached to the bottle.
Types of Culture media.
The chance of detecting scanty or exacting bacteria will be maximized if a variety of rich media are seeded from each sample and different media are used for aerobes and anaerobes. But use of a single, good all-purpose medium will give nearly as many posi¬tive results, saves in labor and cost of materials, and reduces the risk of confusion in the issue and use of the culture bottles.
Traditional Bottles
-If a single, all-purpose medium is to be used, it should be richly nutritive and capable of supporting the growth of both strict aerobes and strict anaerobes when incubated sealed and unexposed to an external atmosphere. A good choice is a Robertson-type cooked-meat medium based on brain heart infusion broth and having at its foot a layer of pieces of lean meat about 3 cm deep . The inoculated blood adds nutrients, including X and V factors, and small amounts of O2 and CO2. When the bottle is incubated while still sealed, most anaerobes will find suitable conditions for growth among the pieces of meat and strict aerobes will grow at the surface of the broth where they have access to oxygen in the overlying air. If the bottle has been autoclaved with its cap tightly closed, its original content of air will still be present. If it has been autoclaved with its cap loose, the air expelled during autoclaving will have been replaced by air drawn into during the subsequent cooling, unless the washer sucked tightly on to its mouth.
If two bottles have been seeded from the sample, their caps may be loosened and one of them incubated in air plus 5-10% CO2, the other in an anaerobic jar, but the advantages of this procedure over that of incubating both bottles still sealed have not been proven.
When the presence of certain exacting organisms is indicated, specially suitable media and methods of incubation should be used Sabouraud broth incubated at 28°C for up to 10 days for yeasts and fungi.
Recent Blood Culture Bottles
-Casteneda-rvpe bottles. The difficulties of making many repeated examinations of blood cultures may be avoided by the use of bottles of the Casteneda type, which contain an agar surface above the level of the culture fluid. Repeated attempts at subculture may be made with a minimum of labor and without ever opening the bottle and exposing it to the risk of contamination.
The bottle is momentarily tilted to run some culture fluid over the agar surface and then re-incubated until colonies are seen to appear on the latter.
-A commercially available kit (Roche) supplies the agar medium in a clear plastic container that is attachable to the mouth of a conventional blood culture bottle on its receipt in the laboratory.
-Another kit (GIbco) contains the agar slope in a parallel chamber of a double bottle into which some of the seeded broth can be tipped.
-Gas capture system. A simple blood culture system recently developed by Oxoid obviates the need for expensive automated equipment, demonstrates both aerobic and anaerobic organisms in a single culture bottle, and permits frequent, easy observation of the onset of growth. The blood culture bottle contains a medium specially formulated to promote gas production on the growth of any aerobic or anaerobic organism and the gas pressure forces medium into a reservoir where it can be observed visually in the course of incubation.
Automated systems
These systems employ equipment that automatically detects an early sign of bacterial growth in a special blood culture bottle. The system most used is Bactec (Becton Dickinson).
It depends on the release of carbon dioxide (C02) into the atmosphere in the culture bottle by the bacterial degradation nutrients in a special culture medium and PH of the bottle becomes acidic leading to release of fluorescent signal detected by specific sensor in Bactec system.
Separate bottles of special medium are supplied for aerobic and anaerobic culture, and bottles of medium containing resins to inactivate any antibiotics that may be present in the specimen.
Normally 10 ml blood would be collected in a syringe and up to 5 ml injected through their rubber caps into aerobic and anaerobic culture. bottles containing 30 ml medium. Up to 60 bottles of the one kind seeded from different patients are loaded into an instrument in which they are incubated, agitated and periodically flushed with an appropriate gas (air + CO2, or N2 + H2 + C02) through two heat-sterilized needles inserted automatically through the rubber cap). It can indicate early bacterial growth, which is often detectable on the same day as the specimen is received in the laboratory.
When growth is thus first detected, the bottle is removed and examined in the usual way by filming and subculture. Negatively reacting bottles do not need to be filmed or subcultured at any stage.